Design and synthesis and testing of conformationally constrained peptidomimetics.

Abstract

This thesis describes the design, synthesis and testing of peptidomimetics pre-organised into bioactive conformations. Chapter One introduces the concept of peptidomimetics, their importance as potential pharmaceuticals. The concept of constraining a compound into a bioactive conformation (α-helix, β-turn or β-strand) by incorporation of a ring or bridge is discussed. The technique of ring closing metathesis as a strategy for cyclisation of peptidomimetics is introduced. Chapter Two surveys β-turn mimics comprised of β-amino acids. The synthesis of novel cyclic peptidomimetics comprised of β-amino acids (cyclised by ring closing metathesis) is presented. Three of the cyclic dipeptides were predicted (through in silico conformational searches) to adopt a β-turn motif. Cyclic scaffolds 2.62, 2.65 and 2.66 were each incorporated into a tri-peptide to give 2.68, 2.69 and 2.70. The propensity of each tri-peptide to adopt a β-turn motif was investigated by 1H NMR. There is strong evidence that 2.70 has a β-turn geometry based on the presence of an intra-molecular hydrogen bond between the i and i+3 residues. Chapter Three introduces cysteine protease calpain II as the primary biological target for this thesis. Calpain is implicated in cataract formation and its inhibition is a logical approach to cataract prevention. Proteases are known to, almost universally, bind substrates and inhibitors in a β-strand conformation. Four macrocycles, designed to be preorganised in a β-strand geometry, were synthesised by ring closing metathesis (compounds 3.02 – 3.05). Macrocycle 3.02 was made to investigate the suitability of an N-terminal 4-fluoro-benzyl-sulfonyl (FBS) in macrocyclic calpain inhibitors. The synthesis of 3.02 was optimised to give the required compound in 33% yield compared to a reported 1% for analogue CAT0811. Diols 3.03 and 3.04 (as a mixture with 3.03 in an 85:15 ratio) were designed to explore possible hydrophilic interactions with the active site of calpain. Macrocycle 3.05 was designed to investigate the relative importance of having an aromatic residue at P₁ for inhibition of calpain, α-chymotrypsin and the 20S proteasome. Chapter Four reports the in vitro testing of macrocycles 3.02, 3.03 and 3.04 against calpain II and discusses these results in the context of the SAR study completed by the Abell group to identify the criteria for the most potent macrocyclic calpain inhibitor. CAT0811 was confirmed as the most potent macrocyclic calpain II inhibitor to date with an IC₅₀ of 0.03 μM. Macrocycle 3.02 had an IC₅₀ of 0.045 μM against calpain II, confirming the suitability of FBS as an N-terminus in these macrocycles. Macrocycle 3.03 had an IC₅₀ of 3.7 μM against calpain II, suggesting a diol substituent is not tolerated by the enzyme at P₁. Diol 3.04 (as a mixture with 3.03 in an 85:15 ratio) was essentially inactive against calpain II (IC50 > 50 μM), presumably as 3.04 has a low propensity to adopt a β-strand conformation. Macrocycle 3.05 had an IC₅₀ of 0.15 μM against calpain II, a Ki of 686 μM against α-chymotrypsin and an IC₅₀ of 1.46 μM against the chymotrypsin-like sub-site of the 20S proteasome. CAT0811 was inactive against α-chymotrypsin and had an IC₅₀ of 1.51 μM against the chymotrypsin-like sub-site of the 20S proteasome. While modification to the P₁ and P₃ positions moderately influenced the selectivity of the macrocycles (comparing 3.05 with CAT0811), a much more dramatic affect was gain by modification of the P₂ residue (as in 4.02). Chapter Five reports the synthesis and in vivo testing of tritiated analogues of CAT0811 and 4.04 by reduction of CAT0811 with NaBT₄ to give macrocycle 5.02, and subsequent oxidation of 5.02 to give 5.03. Compounds 5.02 and 5.03 were separately formulated and administered to sheep from the cataract flock. Liquid scintillation counting was used to get a preliminary outlook on the absorption, distribution and excretion of the macrocycles and to investigate the phenomenon of les crossover of the inhibitors. Previous in vivo trials of CAT0811 have reported that when the formulated inhibitor is administered to the left lens, both lenses are equally observed to have slowing of cataract progression (p < 0.05). Levels of tritium in the treated and untreated lenses were measured. Equal amounts of 5.02 were found in both lenses 48 h after application. This supports our hypothesis that lens crossover of the macrocycles is occurring.